Isolation of Plastids from Sunflower Cotyledons during Germination

132

A technique for the isolation of intact plastids from spinach (Spinacia oleracea) and pea (Pisum sutivum) leaves, pea roots and castor bean (Ricinus communis) endosperm is described. This technique involves brief centrifugation of whole homogenates on-density gradients. Intact plastids were located in the gradient by assaying for triose phosphate isomerase activity. Contamination of the plastic peak with mitochondria and microbodies was estimated by measurement of cytochrome otidase and catalase, respectively. For three of the four tissues the level of toontamination of the plastids by these organelles was 2% or less. The sedimentation behavior of microbodies from different tissues is discussed.Various workers have isolated intact plastids by aqueous means from a range of tissues (13,15,20,21,23,24,27). In many of these examples, their isolation and purity was monitored only by electron microscopy. This method allows the positive conclusion that intact plastids have been isolated to a reasonable degree of purity but does not readily allow an estimate to be made of the yield or the degree of contamination with other organelles. Often these studies have said little about the enzyme complement of the intact plastids.Studies in which the enzyme levels of the plastids and other organelles have been measured after separation have been carried out chiefly using sucrose density gradients. In these separations, the band containing intact plastids is highly contaminated with other organelles. The microbodies are the usual source of contamination because of the closeness of their equilibrium density to that of the plastids. This is particularly so in certain stages of development of sunflower cotyledons and results in about 90% cross contamination of plastids and microbodies on the gradients (22). In other tissues (e.g., spinach leaf), the mitochondria and chloroplasts apparently have the same equilibrium density on sucrose gradients (21). Rocha and Ting (21) have attempted to overcome this by combining sedimentation velocity and isopycnic density gradient centrifugation in sequence to separate the organelles. Although this was largely successful for mitochondria and microbodies, the intact plastids had substantial (13%) contamination with mitochondria, and the yield was very small.The fact that the other organelles have equilibrium densities close to that of the plastids means that isopycnic density gradi-

mentioning

confidence: 99%

Isolation of Intact Plastids from a Range of Plant Tissues

Miflin¹,

Beevers²

1974

132

“…Ic). Triose-P isomerase has been shown to occur in etioplasts and in the cytosol (21). Some catalase was associated with the mitochondrial fraction (Fig.…”

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confidence: 99%

Subcellular Localization of Hexose Kinases in Pea Stems: Mitochondrial Hexokinase

Tanner¹,

Copeland²,

Turner³

1983

The subcellular localization of hexose phosphorylating activity in extracts of pea stems has been studied by differential centrifugation and sucrose density gradient centrifugation. The hexokinase (EC 2.7.1.1) was associated with the mitochondria, whereas fructokinase (EC 2.7.1.4) was in the cytosolic fraction. Some properties of the mitochondrial hexokinase were studied. The enzyme had a high affinity for glucose (Km 76 micromolar) and mannose (Km 71 micromolar) and a relatively low affinity for fructose (Km 15.7 millimolar). The Km for MgATP was 180 micromolar. The addition of salts stimulated the activity of the hexokinase. Al3" was a strong inhibitor at pH 7 but not at the optimum pH (8.2). The enzyme was not readily solubilized but, in experiments with intact mitochondria, was susceptible to proteolysis. A location on the outer mitochondrial membrane is suggested for the hexokinase of pea stems.The phosphorylation of hexoses by ATP is usually the first reaction in the metabolism of these sugars. Two isoenzymes of hexokinase (ATP: D-hexose 6-Ptransferase, EC 2.7.1.1) have been characterized in yeast (4,18). Mammalian tissues contain four forms of hexokinase which have been designated I, II, III, and IV in order of elution from a DEAE-cellulose column (4, 18). Hexokinases I and II, the predominant forms in brain and skeletal muscle, respectively, bind reversibly to mitochondria and are readily solubilized by glucose-6-P (20). It is thought that the bound hexokinase is the more active form (15). Hexokinase III does not associate with the particulate fraction (14).Plant tissues contain several enzymes capable of phosphorylating hexoses. In mature pea seeds, there are two hexokinases and two fructokinases (ATP: D-fructose 6-Ptransferase, EC 2.7.1.4) (24): these have been termed hexokinase I, hexokinase II, fructokinase I, and fructokinase II. It has been suggested that hexokinases I and II are primarily involved in the phosphorylation of glucose and mannose, whereas fructokinase I and II are largely responsible for the phosphorylation of fructose (6,(24)(25)(26).There is little information available on the intracellular distribution of the hexose kinases in plant tissues. In the present investigation, the subcellular localization of hexokinase and fructokinase in pea stems has been studied. Pea stems were used as they are more suited to subcellular fractionation than pea seeds. The hexokinase of pea stems was associated with the mitochondria, whereas the fructokinase was soluble. Subcellular Fractionation of Pea Stem Extracts. Pea seeds were surface-sterilized for 10 min in 0.7% (w/v) NaOCl, soaked in water for 5 h, and germinated in Perlite in the dark for 10 d at 20°C. The seedlings were chilled at 4°C, the apical hook removed, and the stems harvested and rinsed in distilled H20. All subsequent operations were carried out at 4°C. Chilled pea stems (100 g) were sliced into 2-cm lengths and suspended in an equal volume of 1 mm Tes-KOH buffer (pH 7.2) which contained 0.3 M mannitol, 1 mm EDTA, and 1 mm G...

“…Mingren) seeds were soaked overnight in water and germinated in moist paper toweling for 6 days in the dark. The cotyledons were prepared at 4 C under a nitrogen atmosphere by chopping 10 g of the cotyledons in an onion chopper with 20 ml of a medium containing 0.165 M Tricine buffer, pH 7.5; 10 mm EDTA; 10 mM dithioerythritol; 10 mm KCl; 10 mM MgCI2; and 1.0 M sucrose (7). The mixture was transferred to a mortar and ground gently under N, for 2 min with a pestle.…”

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confidence: 99%

Intracellular Distribution of Hydroperoxide Isomerase

Zimmerman

Vick²,

Borg³

1974

Differential centrifugation of several plant extracts indicates that the majority of the hydroperoxide isomerase activity is present in the cytoplasm of the cell. However, lesser amounts of isomerase activity were found in the mitochondrial and microsomal fractions of sunflower seedlings. Sucrose density gradient centrifugation of extracts from sunflower, watermelon, and flax seedlings and from cauliflower buds showed that isomerase activity was associated with the mitochondria. There was no evidence for presence of hydroperoxide isomerase activity in the microbodies.The identification of a flaxseed hydroperoxide isomerase enzyme was first reported in 1966 (12). This enzyme converts the hydroperoxides resulting from the action of lipoxygenase with linoleic or linolenic acids to the corresponding a-ketol and has since been identified in many plant tissues (2,3,8,10,11,13). To determine the functional role of the hydroperoxide isomerase enzyme in plant metabolism, we have investigated the intracellular location of the enzyme. This paper reports the presence of hydroperoxide isomerase activity in mitochondria isolated from sunflower cotyledons and seeds by sucrose density gradient centrifugation. In addition, the paper reports the presence of this activity in the mitochondria of watermelon, flax, and cauliflower. MATERIALS AND METHODSContinuous, concave sucrose density gradients ranging from 33% (w/w) sucrose to 60% sucrose were prepared over a 3-ml base of 60% sucrose in 7.5-X 2.5-cm centrifuge tubes. The sucrose gradient contained 1 mm EDTA.Sunflower (Helianthus annuus L. var. Mingren) seeds were soaked overnight in water and germinated in moist paper toweling for 6 days in the dark. The cotyledons were prepared at 4 C under a nitrogen atmosphere by chopping 10 g of the cotyledons in an onion chopper with 20 ml of a medium containing 0.165 M Tricine buffer, pH 7.5; 10 mm EDTA; 10 mM dithioerythritol; 10 mm KCl; 10 mM MgCI2; and 1.0 M sucrose (7). The mixture was transferred to a mortar and ground gently under N, for 2 min with a pestle. The for 10 min to remove cellular debris, and the supernatant was centrifuged at 10,000g for 20 min. The resulting pellet was resuspended in 1 ml of the homogenizing medium. Large clumps of the pellet were broken by repeated withdrawal into a 2-ml volumetric pipette. The suspension was then layered on the sucrose density gradient and centrifuged 5 hr at 58,000g. For differential centrifugation, the 480g supernatant was centrifuged at 50,000g for 20 min, and the pellet was washed twice with homogenizing medium. The first 50,000g supernatant was centrifuged at 105,000g for 1 hr, and the pellet was washed twice with homogenizing medium.Mitochondria were isolated from ungerminated sunflower (Helianthus annuus L. var. Mingren) seeds by grinding 260 g of the seeds, with hulls removed, for 10 sec in a high speed grinder. The resulting meal was defatted by washing with 1300 ml of cold petroleum ether in a Buchner funnel at 4 C and placed in a vacuum desiccator to remove the petr...